Microalgae

as an ingredient in aquafeeds
Inclusion of microalgae in diets for gilthead
sea bream (Sparus aurata L.) juveniles
and the effect on intestinal functionality
by Antonio Vizcano, Mara Isabel Sez, Toms Martnez
and Javier Alarcn, LifeBioencapsulation SL Almera, Spain

ishmeal is currently the main

source of protein utilised in
carnivorous fish feeding but
overexploitation of fisheries
combined with growing demand has caused
its price to rise continuously. Consequently,
future expansion of aquaculture
production based on the use of fishmeal
as a major ingredient for aquafeeds will be
unsustainable. Plant protein sources such as
soybean meal, rapeseed meal, corn gluten
meal or wheat gluten are commonly used
as ingredients in commercial aquafeeds.
However such protein ingredients often
contain anti-nutritional factors that can
have negative effects on the digestive tract
of fish (Santigosa et al., 2008; Merrifield et
al., 2009).

Microalgae as an ingredient
in aquafeeds

In this regard, and owing to their

chemical composition, microalgae appear
as a promising alternative for enhancing the
nutritive value of conventional feeds and
for use, at least partially, as a substitute for
fishmeal (Lupatch, 2009).
In general microalgae contain a protein
level ranging from 30-55 percent DM;
about 13-33 percent DM total lipids, with
considerable amounts of highly unsaturated
fatty acids (HUFA) which are indispensable in
fish nutrition; and high vitamin content. They
are also rich in pigments such as chlorophylls,
carotenoids and phycobiliproteins (Vizcaino
et al., 2014). Microalgae therefore stand out
as a promising protein source for aquaculture
and thus might well reduce the ecological
impacts associated with current fishmealbased intensive fish farming if sufficient
quantities of algal biomass become available
at a suitable price (Shields and Lupatch,
2012).

Uses of Scenedesmus
almeriensis microalgae in fish

In a recent study we evaluated the effect

of inclusion of Scenedesmus almeriensis
microalgae as a dietary ingredient on intestinal
proteolytic activity of juvenile sea bream.
Scenedesmus is a native microalga and it
was chosen in the present study owing to its
high protein content, fatty acid profile with
substantial amounts of linolenic acid, 18:3(n3), and remarkable productivity. Biomass was
obtained from the facilities at the Estacin
Experimental Las PalmerillasFundacin
Cajamar (Almera, Spain). The Scenedesmus
alga was cultivated in a large-scale tubular
photobioreactor. The algal biomass was
harvested using a RINA continuous centrifuge
(Riera Nadeu SA, Spain) then frozen,
freeze-dried and finally milled to obtain a
homogenised powder (<100 m) that was
stored in the dark at 20 C until use.
Dry algal biomass was incorporated
into four experimental diets (40 percent
crude protein and 10 percent crude lipid) at
increasing levels (12, 20, 25 and 39 percent).
A feed without Scenedesmus served as
a control diet. Feeds were made at the
University of Almeria-CEIA3 facilities (Service
of Experimental Diets; http://www.ual.es/
stecnicos_spe). Every experimental feed was
randomly assigned to triplicate groups of
fifteen sea bream juveniles (8.0 g initial body
weight). Fish were fed by hand twice per
day (9:00 and 13:00) at a rate of 3 percent
of their body weight over 45 days. At the
end of the trial, fish were killed according
to the requirements of the Directive
2010/63/UE, and the digestive tract was
removed and processed to obtain enzymatic
extracts. Digestive enzymes activity levels
were differentiated in two groups: total
alkaline protease, trypsin, chymotrypsin
and -amylase activity levels were used as
indicators of digestive capacity, while leucine

10 | INTERNATIONAL AQUAFEED | January-February 2015

aminopeptidase and alkaline phosphatase

activity levels were used as indicators of
intestinal absorptive capacity.
In order to visualise the active proteases
present in intestinal extracts of fish fed on
different experimental feeds, substrate-SDS-

Figure 1: Digestive enzyme activities

trypsin (A), L-aminopeptidase (B) and
alkaline phosphatase (C) measured
in intestinal extracts of juvenile sea
bream at the end of feeding trial.
Dietary codes: control, SC12: 12
percent Scenedesmus meal inclusion,
SC20: 20 percent Scenedesmus
meal inclusion, SC25: 25 percent
Scenedesmus meal inclusion, SC39: 39
percent Scenedesmus meal inclusion.
Values are expressed as mean SD
(n=12). Different lowercase letters
indicate significant differences among
experimental groups (P < 0.05).

FEATURE

Figure 2: Zymograms of alkaline

proteolytic activity in intestine extracts
of juvenile S. aurata fed on diets with
increasing Scenedesmus meal levels. All
lanes in zymograms contain the same
enzyme activity (2 units of activity per
well). Dietary codes: control, SC12: 12
percent Scenedesmus meal inclusion,
SC20: 20 percent Scenedesmus
meal inclusion, SC25: 25 percent
Scenedesmus meal inclusion, SC39: 39
percent Scenedesmus meal inclusion.

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Hatchery
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studiedConal
by transmission
microscopy
PAGE
electrophoresis
gels were carried
of SA on digestive enzyme
(TEM),
giving
information
about
the
length
and
out
(Alarcn
et
al.,
1998).
In
addition,
the
activities of sea bream
ernational Aquafeed | Size: Half Page | Dimensions: 190mm X 132mm
presence of protease inhibitors in SA was
tested according to Alarcn et al. (1999).
Possible impacts of the different diets on
the ultrastructure of the intestinal mucosa were

diameter of the microvilli. The intestines of

three fish from each tank were collected for
examination and TEM samples were prepared
according to Vizcaino et al. (2014).

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The ability of fish to use the ingested

nutrients depends on the presence of an
adequate set of digestive enzymes. Hence,
knowledge on nutrient hydrolysis in the

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FEATURE
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digestive tract is an important factor in optimising fish feeds

(Deguara et al., 2003). Intestinal enzymes are correlated
with the nutritional status of fish, and their activities
have been used as indicators of the digestive capacity of
farmed fish (Alarcn et al., 1998). In fact fish are capable
of modulating their digestive enzyme pattern in response
to the feed source, quality and concentration of dietary
nutrients (Santigosa et al., 2008).
The use of Scenedesmus meal to replace fishmeal
protein did not decrease the enzyme activities secreted
into the intestinal lumen at any of the inclusion levels
tested, albeit increased activities attributable to algae
inclusion have been observed. For trypsin activity,
which plays a decisive role in dietary protein hydrolysis
and zymogen activation, fish fed on SC12 showed
significantly higher activity than those animals fed on a
microalgae-free diet. In addition, alkaline phosphatase and
L-aminopeptidase activities increased significantly in the
SC20 group compared to the Control and SC39 groups
(Fig 1). Nevertheless, total alkaline protease, chymotrypsin
and -amylase activities remained unaffected by
Scenedesmus inclusion.
Zymograms carried out on intestinal extracts obtained

12 | INTERNATIONAL AQUAFEED | January-February 2015

FEATURE

Figure 3: Comparative TEM micrographs from the anterior (A) and posterior (B)
intestinal regions of juvenile S. aurata at the end of the feeding trial. Scenedesmusfed fish (SC20 and SC39) showed longer microvilli than control-fed fish. Dietary
codes: CT: control, SC12: 12 percent Scenedesmus meal inclusion, SC20: 20 percent
Scenedesmus meal inclusion, SC25: 25 percent Scenedesmus meal inclusion, C39:
39 percent Scenedesmus meal inclusion (TEM bar: 2 m).
after electrophoretic separation of proteins
are shown in Fig 2. The profile of the
intestinal proteases seems not to be
influenced by the inclusion of Scenedesmus
biomass, given that all the animals showed
the same number and distribution of active
fractions as control-fed fish, characterised by
five groups of active bands.
Another important aspect to consider
when using ingredients alternative to
fishmeal is the presence of anti-nutritive
factors that might interfere with nutrient
digestion and absorption (Alarcn et al.,
1999). Among the wide range of such
factors, protease inhibitors are well known

as substances that can affect dietary

protein utilisation. In this study, neither
Scenedesmus meal (unlikely other protein
sources) nor experimental feeds contained
substances able to inhibit the digestive
proteases of gilthead sea bream juveniles,
given that inhibition never reached more
than 5 percent (data not shown).

Checking effects by histological

study of the intestine

In addition to digestive enzyme activities,

the structure and morphology of the
intestinal mucosa play a key role in nutrient
absorption. TEM analysis of anterior

and posterior intestine (Fig. 3a and 3b,

respectively) revealed that the inclusion of
microalgae reduced microvilli length (ML),
except for the SC20 group (with higher
and similar ML values compared to those of
the CT group in the anterior and posterior
intestine, respectively) and the SC39 group
(with similar and higher ML values compared
to those of control fish in the anterior and
posterior intestine, respectively), and that
microvilli diameter in the anterior intestine
was greater in Scenedesmus-fed fish. Thus,
the overall effects of both results were
increased absorptive surface and improved
contribution of the intestinal mucosa as a
physical barrier.

Conclusions

The inclusion of S. almeriensis increased

the level of intestinal enzyme activities as
well as the intestinal absorptive surface.
Therefore, the combination of these effects,
together with the lack of anti-nutritional
factors, confirms that Scenedesmus biomass
can be used as a dietary ingredient for
juvenile sea bream diets, and furthermore,
that an inclusion level of 20 percent is
recommended according to the positive
effects observed on gut functionality at such
a ratio.

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